Development and optimisation of direct-PCR of SSRs in Elaeis guineensis using QIAcard FTA classic

The genetic studies in oil palm (Elaeis guineensis Jacq.) rely heavily on the extraction of high-quality DNA, typically obtained from young leaves. High-quality of DNA is essential for further downstream applications, particularly PCR-based amplification of simple sequence repeat (SSR). Obtaining young leaf tissues often inaccessible that leaves mature oil palm leaves as an option for DNA source. However, mature leaves are tough and rich in secondary metabolites, which often inhibit PCR reactions and reduce DNA quality. These factors can lead to PCR amplification failure. This study aims to develop an optimised direct-PCR approach using Flinders Technology Associates (FTA) card for mature oil palm leaves, thereby eliminating the need for conventional DNA extraction and purification. A total of 50 mature oil palm leaves were collected and deposited onto FTA cards. Sixteen combinations of FTA card preparation consists of four critical FTA preparation parameters were tested to optimise and develop the direct-PCR method, incorporating primer mEgCIR3607 as preliminary validation primer. The optimised and developed protocol was then validated using nine pairs of SSR primers with high polymorphic information content (PIC). SSR profiling was further conducted across all 50 oil palm samples to demonstrate the applicability of the direct-PCR method. Further study was conducted to validate the FTA matrices using different leaf storage conditions: (1) fresh, (2) frozen at - 20 °C, and (3) deep freeze at -80 °C; as well as different FTA disc coverage consisting of green extract (¼, ½, ¾, and full), stored at both 24 hours and seven days storage period. FTA elute was developed as final product for FTA washing step when using oil palm as DNA source. Combination of centrifugation at 2500 rpm with clear clarity of vortexing, incubate at 95°C for 5 minutes and eluted in 80 μl of TE buffer shows to have a good combination of FTA card preparation modification as the it has high intensity of PCR product with very minimal primer-dimer formation and PCR inhibition that meet the expected amplicon size. SSR profiling demonstrates a consistent monomorphic pattern indicating a similar genetic profile among the assessed individuals. Validation of FTA matrices showed FTA disc coverage and storage period do not influence the PCR amplification, except when using ¼ FTA disc coverage stored at -20°C for 7 days. The results confirm the feasibility of this optimised direct-PCR method for mature oil palm leaves, offering advantages in speed, simplicity, cost-effectiveness, and sustainability for large-scale genetic studies.